1. Summary of the Invention
The present invention relates to novel amides which are amino acid substituted 4-aminophenazones used as substrates for measuring enzyme activity. In particular the present invention relates to amino acid substituted 4-aminophenazones, particularly glutamyl-4-aminophenazones, wherein the amino acid substituent is cleaved by the enzyme which recognizes the amino acid to produce 4-aminophenazone which is then used as an amine dye intermediate which is reacted with a second dye intermediate in the presence of an oxidizing agent to produce a chromogen.
2. Prior Art
Serum Gamma glutamyltransferase (GGT) is elevated in all liver diseases (Moss, D. W., et al. Enzymes. In: Teitz, N. W., ed. Textbook of Clinical Chemistry. Philadelphia: W. B. Saunders, 5:721-722 (1986)). For the detection of obstructive jaundice, cholangitis, and cholecystitis, it is more sensitive than alkaline phosphatase, 5'-nucleotidase, leucine aminopeptidase, and the transaminases (Moss, D. W., et al. Enzymes. In: Teitz, N. W., ed. Textbook of Clinical Chemistry. Philadelphia: W. B. Saunders, 5:721-722 (1986)). Its rise occurs earlier than with these other enzymes and persists longer. The measurement of serum GGT activities is very valuable in the detection of alcohol-induced liver diseases. Not only are elevated levels of GGT found in the sera of patients with alcoholic cirrhosis, but also in the majority of sera from persons who are heavy drinkers (Moss, D. W., et al. Enzymes. In: Teitz, N. W., ed. Textbook of Clinical Chemistry. Philadelphia: W. B. Saunders, 5:721-722 (1986)).
The first methods for GGT measurement involved the use of the physiological substrate glutathione (Hanes, C. S., et al. Biochem. J. 51:25-35(1952); but these were cumbersome and quickly replaced by methods using synthetic substrates. The use of synthetic substrates such as N-(DL-gamma-glutamyl)aniline (Goldbarg, J., et al., Arch. Biochem. Biophys. 91:61-70 (1960)), L-gamma-glutamyl-naphthylamide (Orlowski, M., et al., Clin. Chem. Acta 7:755-760 (1962), and L-gamma-glutamyl-p-nitroanilide (Szasz, G., Clin. Chem. 15:124-136 (1969)) all involve production of dyes from cleavage products, but have definite limitations for routine use. See also Beaty et al U.S. Pat. No. 4,511,651 and Beck et al U.S. Pat. No. 4,567,138.
At present, the substrate of choice for measurement of GGT activity is L-gamma-glutamyl-3-carboxy-4-nitroanilide (gluCANA) (IFCC Methods for the Measurement of Catalytic Concentration of Enzymes, Part 4, IFCC Method for gamma-Glutamyltransferase [(gamma-Glutamyl)-Peptide: Amino Acid gamma-Glutamyltransferase, EC 2.3.2.2], 1983). Its high turnover rate and solubility give it a distinct advantage over its predecesser, L-gamma-glutamyl-p-nitroanilide (gluPA), which suffers from poor solubility. But both substrates have a low molar absorptivity, and both exhibit overlapping spectra of substrate and product, causing measurement to be performed on the spectrum shoulder rather than at the dye's absorption peak (Fossati, P., et al. Clin. Chem., 32:1581-1584 (1986)).